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The quest for HIV-1 cure could take advantage of the study of rare individuals that control viral replication spontaneously (elite controllers) or after an initial course of antiretroviral therapy (posttreatment controllers, PTCs). In this review, we will compare back-to-back the immunological and virological features underlying viral suppression in elite controllers and PTCs, and explore their possible contributions to the HIV-1 cure research.HIV-1 control in elite controllers shows hallmarks of an effective antiviral response, favored by genetic background and possibly associated to residual immune activation. The immune pressure in elite controllers might select against actively transcribing intact proviruses, allowing the persistence of a small and poorly inducible reservoir. Evidence on PTCs is less abundant but preliminary data suggest that antiviral immune responses may be less pronounced. Therefore, these patients may rely on distinct mechanisms, not completely elucidated to date, suppressing HIV-1 transcription and replication.PTCs and elite controllers may control HIV replication using distinct pathways, the elucidation of which may contribute to design future interventional strategies aiming to achieve a functional cure.

A better understanding of the immunological markers associated with long-lasting immune responses to SARS-CoV-2 infection is of paramount importance. In the present study, we characterized SARS-CoV-2-specific humoral responses in hospitalized (ICU and non-ICU) and non-hospitalized individuals at six months post-onset of symptoms (POS) (N = 95). We showed that the proportion of individuals with detectable anti-SARS-CoV-2 IgG or neutralizing (NAb) responses and the titers of antibodies were significantly reduced in non-hospitalized individuals, compared to ICU- or non-ICU-hospitalized individuals at 6 months POS. Interestingly, SARS-CoV-2-specific memory B cells persist at 6 months POS in both ICU and non-ICU patients and were enriched in cells harboring an activated and/or exhausted phenotype. The frequency/phenotype of SARS-CoV-2-specific memory B cells and the magnitude of IgG or NAb responses at 6 months POS correlated with the serum immune signature detected at patient admission. In particular, the serum levels of CXCL13, IL-1RA, and G-CSF directly correlated with the frequency of Spike-specific B cells and the magnitude of Spike-specific IgG or NAb, while the serum levels of CXCL12 showed an antagonizing effect. Our results indicate that the balance between CXCL12 and CXCL13 is an early marker associated with the magnitude and the quality of the SARS-CoV-2 humoral memory.

Purpose of review HIV persists in distinct cellular and anatomical compartments in the body including blood, Central nervous system, and lymphoid tissues (spleen, lymph nodes [LNs], gut-associated lymphoid tissue) by diverse mechanisms despite antiretroviral therapy. Within LNs, human and animal studies have highlighted that a specific CD4 T cell subset - called T follicular helper cells locating in B cell follicles is enriched in cells containing replication-competent HIV as compared to extra-follicular CD4 T cells. Therefore, the objective of the present review is to focus on the potential mechanisms allowing HIV to persist within LN microenvironment. Recent findings The combination of factors that might be involved in the regulation of HIV persistence within LNs remain to be fully identified but may include - the level of activation, antiretroviral drug concentrations, presence of cytolytic mechanisms and/or regulatory cells, in addition to cell survival and proliferation propensity which would ultimately determine the fate of HIV-infected cells within LN tissue areas. Summary HIV persistence in blood and distinct body compartments despite long-standing and potent therapy is one of the major barriers to a cure. Given that the HIV reservoir is established early and is highly complex based on composition, viral diversity, distribution, replication competence, migration dynamics across the human body and possible compartmentalization in specific tissues, combinatorial therapeutic approaches are needed that may synergize to target multiple viral reservoirs to achieve a cure for HIV infection.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

During the last phase of HIV viral production, nascent HIV virions acquire a fraction of the cellular lipid membrane to create the external lipid envelope, a process by which cellular proteins present on the surface of the infected cell can be incorporated along with Env trimers. Interestingly, several studies indicated that these incorporated host molecules could conserve their biological activity and consequently contribute to HIV pathogenesis either by enhancing the infectivity of HIV virions, their tissue tropism or by affecting immune cell functions. The following review will describe the main approaches used to characterize membrane bound host molecule incorporation into HIV virions, the proposed mechanisms involved, and the role of a non-exhaustive list of incorporated molecules.

PURPOSE OF REVIEW Cell-based immunotherapies have made enormous progress over the last decade with the approval of several anti-CD19-chimeric antigen receptor (CAR)-T cell therapies for haemato-oncological diseases. CARs are synthetic receptors comprising an antigen-specific extracellular domain fused to a hinge, transmembrane and intracellular signalling domains. The success obtained with CD19 CAR-T cells rekindled interest in using CAR-T cells to treat HIV seropositive patients. The purpose of this review is to discuss historical and recent developments of anti-HIV CARs. RECENT FINDINGS Since the first description of CD4+-based CARs in the early 90s, new generations of anti-HIV CARs were developed. They target the hetero-trimeric glycoprotein gp120/gp41 and consist of either a CD4+ extracellular domain or a VH/VL segment derived from broadly neutralizing antibodies. Recent efforts were employed in multiplexing CAR specificities, intracellular signalling domains and T cells resistance to HIV. SUMMARY Several new-anti HIV CAR-T cells were successfully tested in preclinical mice models and are now waiting to be evaluated in clinical trials. One of the key parameters to successfully using CAR-T cells in HIV treatment will depend on their capacity to control the HIV reservoir without causing off-targeting activities.

The majority of cells with latent human immunodeficiency virus 1 infection are located in lymphoid tissues that are difficult to access. In the current study, we used single-genome near-full-length proviral sequencing to evaluate intact and defective proviruses in blood and lymph node CD4 T cells enriched for specific functional polarizations. We observed minor variations between the frequencies of proviral sequences within individual CD4 T-cell subsets and across tissue compartments. However, we noted multiple clonal clusters of identical intact or defective proviral sequences from distinct compartments and CD4 T-cell subpopulations, suggesting frequent interchanges between viral reservoir cells in blood and tissues.

Activation of HIV-1 reservoirs and induction of anti-HIV-1 T cells are critical to control HIV-1 rebound after combined antiretroviral therapy (cART). Here we evaluated in humanized mice (hu-mice) with persistent HIV-1 infection the therapeutic effect of TLR3 agonist and a CD40-targeting HIV-1 vaccine, which consists of a string of 5 highly conserved CD4+ and CD8+ T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol fused to the C-terminus of a recombinant anti-human CD40 antibody (αCD40.HIV5pep). We show that αCD40.HIV5pep vaccination coadministered with poly(I:C) adjuvant induced HIV-1-specific human CD8+ and CD4+ T cell responses in hu-mice. Interestingly, poly(I:C) treatment also reactivated HIV-1 reservoirs. When administrated in therapeutic settings in HIV-1-infected hu-mice under effective cART, αCD40.HIV5pep with poly(I:C) vaccination induced HIV-1-specific CD8+ T cells and reduced the level of cell-associated HIV-1 DNA (or HIV-1 reservoirs) in lymphoid tissues. Most strikingly, the vaccination significantly delayed HIV-1 rebound after cART cessation. In summary, the αCD40.HIV5pep with poly(I:C) vaccination approach both activates replication of HIV-1 reservoirs and enhances the anti-HIV-1 T cell response, leading to a reduced level of cell-associated HIV-1 DNA or reservoirs. Our proof-of-concept study has significant implication for the development of CD40-targeting HIV-1 vaccine to enhance anti-HIV-1 immunity and reduce HIV-1 reservoirs in patients with suppressive cART.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers., Author summary Increasing number of evidences indicate that B-cell follicles might be anatomical sanctuaries for active transcription in both HIV/SIV viremic controllers and in ART treated aviremic HIV-infected individuals. While multiple mechanisms may be involved in the regulation of HIV transcription, recent studies suggested that immune checkpoint molecule (IC) signaling may contribute to maintain HIV-1 latency in infected CD4 T cells. These observations prompted us to investigate the involvement of IC/IC-L interactions in the regulation of HIV-1 transcription in lymph node (LN) tissues. In the present study, we show that T follicular helper (Tfh) cells predominantly co-expressed PD-1 and TIGIT, which were functionally active. An in-depth mass cytometry analysis revealed that PD-L1, PD-L2 (PD-1 ligands) and CD155 (TIGIT-ligand) were predominantly co-expressed on a specific LN dendritic cell (DC) subpopulation expressing markers of migratory DCs. We subsequently demonstrated that LN migratory DCs, locating predominantly in LN extra-follicular areas, could modulate HIV-1 transcription by a mechanism involving PD-L1/PD-1 interactions. Interestingly, the frequency of LN migratory DCs inversely correlated with HIV-1 transcription from LN memory CD4 T cells, suggesting that IC-L expressing migratory DCs might contribute to control HIV-1 transcription and maintain HIV-1 latency in extra-follicular areas. These findings represent a step forward in our understanding of potential mechanisms contributing to the regulation of HIV persistence in lymphoid tissues.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies., The study was funded by the Bill & Melinda Gates Foundation (project nr OPP1040705) and we thank Dr. Pervin Amklesaria and Dr. Nina Russell (Gates Foundation) for their guidance of this study. We thank the Poxvirus T-cell Vaccine Discovery Consortium funded by the Bill & Melinda Gates Foundation (OPP38599) for the development and provision of NYVAC and DNA vectors; support for the ICS and antibody immune monitoring assays was provided by the Vaccine Immune Monitoring Centers (OPP1032325 & OPP1032144), and support for the statistical analysis from the Vaccine Immunology Statistical Center (OPP1032317), all part of the Collaboration for AIDS Vaccine Discovery.

Mycobacterium tuberculosis and human immunodeficiency virus (HIV) infections are coendemic in several regions of the world, and M. tuberculosis/HIV-coinfected individuals are more susceptible to progression to tuberculosis disease. We therefore hypothesized that HIV infection would potentially impair M. tuberculosis-specific protective immunity in individuals suffering from latent tuberculosis infection (LTBI) or active pulmonary tuberculosis (PTB). In this study, we demonstrated that M. tuberculosis/HIV-coinfected individuals have fewer circulating M. tuberculosis-specific CD4 T cells and that those that remained were functionally impaired in both LTBI and PTB settings. In addition, we showed that HIV infection significantly interferes with M. tuberculosis-induced systemic proinflammatory cytokine/chemokine responses. Taken together, these data suggest that HIV infection impairs functionally favorable M. tuberculosis-specific immunity., Human immunodeficiency virus (HIV) infection is the major risk factor predisposing for Mycobacterium tuberculosis progression from latent tuberculosis infection (LTBI) to tuberculosis disease (TB). Since long-term-treated aviremic HIV-infected individuals remained at higher risk of developing TB than HIV-uninfected individuals, we hypothesized that progression from LTBI to pulmonary TB (PTB) might be due not only to CD4 T-cell depletion but also to M. tuberculosis-specific CD4 T-cell functional impairment. To test this hypothesis, M. tuberculosis-specific T-cell frequencies and cytokine profiles were investigated in untreated Tanzanian individuals suffering from LTBI (n = 20) or PTB (n = 67) and compared to those of untreated M. tuberculosis/HIV-coinfected individuals suffering from LTBI (n = 15) or PTB (n = 10). We showed that HIV infection significantly reduced the proportion of Th2 (interleukin 4 [IL-4]/IL-5/IL-13) producing M. tuberculosis-specific CD4 T cells and IL-2-producing M. tuberculosis-specific CD4 and CD8 T cells in individuals with LTBI or PTB (P

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32 + CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1 + CD4 T cells. The frequency of CD32 + CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32 + cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32 + CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32 + and PD-1 + CD4 T cells compared to CD32 - and PD-1 - cells in both viremic and treated individuals, but there was no difference between CD32 + and PD-1 + cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32 + versus PD-1 + cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32 + PD-1 + CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32 - PD-1 - (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32 + PD-1 - (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32 - PD-1 + (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32 + PD-1 - and CD32 - PD-1 + CD4 T cells. Interestingly, the proportion of CD32 + and PD-1 + CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

Age-mixing patterns are of key importance for understanding the dynamics of human immunodeficiency virus (HIV)-epidemics and target public health interventions. We use the densely sampled Swiss HIV Cohort Study (SHCS) resistance database to study the age difference at infection in HIV transmission pairs using phylogenetic methods. In addition, we investigate whether the mean age difference of pairs in the phylogenetic tree is influenced by sampling as well as by additional distance thresholds for including pairs. HIV-1 pol-sequences of 11,922 SHCS patients and approximately 240,000 Los Alamos background sequences were used to build a phylogenetic tree. Using this tree, 100 per cent down to 1 per cent of the tips were sampled repeatedly to generate pruned trees (N = 500 for each sample proportion), of which pairs of SHCS patients were extracted. The mean of the absolute age differences of the pairs, measured as the absolute difference of the birth years, was analyzed with respect to this sample proportion and a distance criterion for inclusion of the pairs. In addition, the transmission groups men having sex with men (MSM), intravenous drug users (IDU), and heterosexuals (HET) were analyzed separately. Considering the tree with all 11,922 SHCS patients, 2,991 pairs could be extracted, with 954 (31.9 per cent) MSM-pairs, 635 (21.2 per cent) HET-pairs, 414 (13.8 per cent) IDU-pairs, and 352 (11.8 per cent) HET/IDU-pairs. For all transmission groups, the age difference at infection was significantly (P

We recently demonstrated that lymph nodes (LNs) PD-1 javax.xml.bind.JAXBElement@6be93b55 /T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1 javax.xml.bind.JAXBElement@21c52b78 CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1 javax.xml.bind.JAXBElement@1041416b CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8(+) T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb-specific CD8(+) T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb-specific CD8(+) T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb-specific CD8(+) T cells in LTBI subjects were mostly T EMRA cells (CD45RA(+) CCR7(-)), coexpressing 2B4 and CD160, and in TB patients were mostly TEM cells (CD45RA(-) CCR7(-)), expressing 2B4 but lacking PD-1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb-specific CD8(+) T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro-expanded Mtb-specific CD8(+) T cells expressed perforin and granulysin. Finally, Mtb-specific CD8(+) T-cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb-specific CD8(+) T-cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb-specific CD8(+) T-cell responses. These results provide new insights in the interaction between Mtb and the host immune response.

Follicular T helper cells are the major reservoir for HIV infection and accumulate during chronic HIV infection., In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5−PD-1−Bcl-6−, CXCR5+PD-1−Bcl-6−, CXCR5−PD-1+Bcl-6−, and CXCR5+PD-1+Bcl-6+. On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5+PD-1+Bcl-6+ cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5−PD-1+ cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (

The persistence of HIV within long-lived HIV-infected CD4 T cells is the primary obstacle towards HIV eradication and numerous strategies are currently being evaluated to target and kill HIV-infected cells to ultimately find a cure. HIV reservoirs are classically quantified by standard methods such as integrated HIV DNA (Alu PCR) and/or quantitative viral outgrowth assay; however, recent technical advances may offer new opportunities to comprehensively assess the impact of clinical interventions.Digital droplet PCR, tat/rev-induced limiting dilution analysis, enhanced quantitative viral outgrowth assay, and whole genome sequencing technologies offer increased precision and/or higher sensitivity to quantify and characterize HIV reservoirs in antiretroviral therapy-treated HIV-infected patients.The objective of this review is to highlight the characteristics and limits of recent technical advances that may help to monitor the impact of clinical interventions in antiretroviral therapy-treated patients.

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4(+) T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4(+) T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry–based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4(+) T cells is a new tool for the rapid diagnosis of active tuberculosis disease.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127+perforin−/CD127−perforin+, and CD127−/perforin−CD8 T cells, respectively. CD127−/perforin−and CD127−/perforin+cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin+/IL-2−or perforin−/IL-2+cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P< 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127+/IL-2-secreting) and cytotoxic (perforin+) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcγ receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC–T cell cocultures. The present results indicate that Ad5 IC activates a DC–T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

Antipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis ofAdenoviridaeimmunogenicity and that each may have specific clinical advantages.

The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro . Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

Background These two trials under Europe-Africa collaborations aim at addressing two factors relevant for Africa i.e helminth infections and HIV-1 diversity. EV06 used a novel combination of DNA expressing clade C Env, Gag and Pol-nef co-administered with AIDSVAX®B/E Env protein to study the effect of S. mansoni on vaccine responses. GREAT is a recently awarded trial using a 2nd generation improved conserved tHIVConsvX T-cell vaccine candidate combined with bivalent mosaic design to increase breadth and protective epitopes. Methods EV06 enrolled 72 males and females aged 18–45, half infected with S. mansoni (SM+). In each arm 30 received vaccine and 6 placebo at week 0, 4 and 24. Responses were evaluated at week 0, 6, 26 and 36. Humoral responses were measured as binding IgG against a panel of HIV-1 envelope glycoproteins and as neutralizing antibodies (Nabs), using TZM/ bl cells and tier 1 pseudoviruses. Cellular responses were measured as HIV-specific CD4+ and CD8+ T-cell by IFN-γ ELIS-pot and multi-cytokine intracellular staining flow cytometry. GREAT will be a phase IIa trial and preparation for efficacy trials in Kenya, Uganda and Zambia testing ChAdOx1.tHIVconsv5 and ChAdOx1.tHIVconsv6 followed by MVA.tHIVconsv3 and MVA.tHIVconsv4 on week 2 (Arm 1) or week 8 (Arm 2). Progress Differences in binding IgG response rates were observed in vaccinated participants against the vaccine matched clade C V1V2 (gp70–96ZM651.02 V1V2) at week 6: 56% among SM+ versus 86% among SM – (p=0.039). At week 36, response magnitudes were statistically lower in the SM+ against gp120 and gp140 proteins (p=0.04 for both). SM+ also had lower Nabs and ELISpot responses at various time points. Still blinded data on the first 20 volunteers show 80% responders for CD4 T cell at w26 and 70% CD8 responders at w36. These trials will provide more data on challenges facing HIV vaccine development in Africa.

PURPOSE OF REVIEW: T follicular helper (Tfh) cells play a critical role as providers of B-cell help and dysfunction in Tfh/B-cell interactions can lead to autoimmunity or immunodeficiency. These observations have generated a great deal of interest in understanding how these cells are affected during HIV infection and how their functional changes might affect antibody responses. RECENT FINDINGS: Recent studies have shown that HIV/simian immunodeficiency virus (SIV) infection affects both Tfh-cell frequency and function and suggest that Tfh-cell perturbations might contribute to the relative inefficiency of HIV-infected individuals to generate broadly neutralizing antibodies (bNAbs). SUMMARY: The present review will highlight these recent findings addressing the role of Tfh cells in HIV infection as well as the impact HIV infection has on Tfh and circulating memory Tfh (cTfh) cell frequency and function.

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160+ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160+ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression., Author Summary T-cell immune response is regulated by a variety of molecules known as co-inhibitory receptors. The over expression of co-inhibitory receptors has been observed in several chronic viral infections such as HIV disease, and is found to be associated with severe T-cell dysfunction. Recent studies have demonstrated that the co-expression of several co-inhibitory receptors correlated with greater impairment of CD8 T cells. However, the relative contribution of individual co-inhibitory receptors to the regulation of T-cell functions remains unclear. In order to shed light on these issues, we have evaluated the influence of the expression of 3 major co-inhibitory receptors such as PD-1, 2B4 and CD160 on CD8 T-cell functions such as proliferation, cytokines production and expression of cytotoxic granules. We demonstrate that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression and that the blockade of CD160 signaling may partially restore CD8 T-cell functions.

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses., Author Summary CD8 T cells directed against virus are complex and functionally heterogeneous. One relevant component of CD8 T cells is their functional avidity which reflects their sensitivity to cognate antigens, i.e. how prone T cells are to respond when they encounter low doses of antigens. In patients with chronic and established HIV infection, we observed that the sensitivity of HIV-specific CD8 T cells was not different between patients with progressive or non-progressive disease. In contrast, the sensitivity of HIV-specific CD8 T cells was significantly lower in patients with early and recent HIV infection. Furthermore, CD8 T cells of high avidity were preferentially associated with a state of functional impairment known as exhaustion. Of interest, some patients treated with antiretroviral therapy during acute infection spontaneously interrupted their treatment and experienced a rebound of virus. In these patients, the avidity of HIV-specific CD8 T cells increased and this increase was associated to stronger cell exhaustion and greater renewal of the population of antiviral CD8 T cells, thus potentially providing the mechanistic basis for the generation of high-avidity CD8 T cells. Overall, our data suggest that rapid perturbation in viremia levels drove increases in the functional avidity of HIV-specific CD8 T cells.

Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.

In the present study, we have investigated the anatomic distribution in blood and gut mucosal tissues of memory poxvirus-specific CD4 and CD8 T cells in subjects vaccinated with smallpox and compared it with vector (NYVAC)-specific and HIV insert-specific T-cell responses induced by an experimental DNA-C/ NYVAC-C vaccine regimen. Smallpox-specific CD4 T-cell responses were present in the blood of 52% of the subjects studied, while smallpox-specific CD8 T cells were rarely detected (12%). With one exception, smallpox-specific T cells were not measurable in gut tissues. Interestingly, NYVAC vector-specific and HIV-specific CD4 and CD8 T-cell responses were detected in almost 100% of the subjects immunized with DNA-C/NYVAC-C in blood and gut tissues. The large majority (83%) of NYVAC-specific CD4 T cells expressed α4β7 integrins and the HIV coreceptor CCR5. These results demonstrate that the experimental DNA-C/NYVAC-C HIV vaccine regimen induces the homing of potentially protective HIV-specific CD4 and CD8 T cells in the gut, the port of entry of HIV and one of the major sites for HIV spreading and the depletion of CD4 T cells.

Methods Virus-specific CD8 T-cell responses (n = 74) including CMV, EBV, Flu and HIV-1 were characterized using tetramer complexes or peptide stimulation and analyzed for proliferation capacity, expression of CD127 and perforin. CCR7, CD45RA, PD-1 and CD57 were used to assess T-cell differentiation/exhaustion. Simultaneous expression of IFNγ, IL-2, TNFα and perforin was analysed by polychromatic flow cytometry.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models., Author Summary In most cases, adenoviruses are thought to initially enter the host via contact with epithelial cells and spread within the host via an unknown mechanism. Most adenovirus serotypes use a cell adhesion molecule dubbed “CAR” to attach to cells. To assess, predict and understand adenovirus biology and vectorology, many in vivo studies use mice and monkeys. These animal models have been considered reliable models in the realm of viral pathogenesis and gene transfer. One of the implications of our study suggests that the rat may be a more appropriate model during intravenous adenovirus delivery because like humans, and unlike mice and monkeys, they also express CAR on their erythrocytes. The identification of CAR on human erythrocytes explains a 50-year-old enigma of adenovirus hemagglutination, helps us better understand adenovirus in vivo biology and may open new avenues to understand the role of cell adhesion molecules during erythropoiesis.

International audience; By the time we are adolescents most of us have been in contact with several of the >50 human adenovirus (HAd) serotypes. These common subclinical infections lead to an efficient anti-adenovirus cross-reacting adaptive immunity. During gene therapy, the ubiquitous anti-adenovirus humoral response and complement activation will modify and dictate vector biodistribution, as well as the response to the virion and transgene(s). In this study, we assayed the interactions of a xenogenic adenovirus derived from canine serotype 2 (CAV-2) with naturally occurring human antibodies (Abs) and the complement system. In our cohort, we found class G immunoglobulins (Igs) that recognized the intact CAV-2 virion and the external virion proteins. However, the majority of donors had low or no neutralizing Abs, class A, or class M Igs. Purified anti-HAd serotype 5 Abs also recognized CAV-2 virion proteins. In addition, in spite of the presence of anti-CAV-2 IgGs, CAV-2 poorly activated the classical and alternative complement cascades. This atypical response was due to a block upstream of the component 3 (C3) convertase and interplay between the component 1 (C1) inhibitor, the C1q-C1r2-C1s2 complex and CAV-2. Our data demonstrate that some xenogenic adenovirus vectors, like CAV-2, could lead to notably different outcomes following systemic delivery.

PURPOSE OF REVIEW Major advances have been made in the delineation of HIV-specific immune response and in the mechanisms of virus escape. The kinetics of the immunological and virological events occurring during primary HIV infection indicate that the establishment of the latent HIV reservoir, the major obstacle to HIV eradication likely occurs during the very early stages of primary infection, that is, the 'eclipse phase', prior to the development of the HIV-specific immune response which has limited efficacy in the control of the early events of infection. Therefore, the window of opportunity to develop effective interventions either to clear HIV during primary infection or to prevent rebound of HIV in patients successfully treated who stop antiretroviral therapy is very narrow. RECENT FINDINGS Genetic factors most strongly associated with nonprogressive infection are human leukocyte antigen (HLA) class I alleles and particularly HLA-B5701. CD4 and CD8 T-cell responses with polyfunctional profile are associated with nonprogressive infection. Broader neutralizing antibodies are detected 3-4 years after infection, generated only in 20% of individuals but show no efficacy in the control of HIV replication. SUMMARY In the present review, we shall discuss the different components of the HIV-specific immune response elicited by the infection, the kinetics of these responses during primary infection and the changes following transition to the chronic phase of infection, and the functional profile of 'effective' versus 'noneffective' HIV-specific immune responses.